This invention specifically relates to chromatography used in biochemical and immunochemical studies.
Chromatography is a technique employed for isolating a biological molecule from a mixture containing possibly hundreds of other contaminating molecules. Isolation of an enzyme from crude cell-free extract is an example. The general requirements for chromatography are a solid phase consisting a matrix with a functional group and a liquid phase containing a buffer for washing and elution. These two phases play intricate role in the success of experiments. Since biological molecules vary from one another in size, shape, charge, composition and function, different types of chromatography procedures are developed based on distinct strategies. For instance, sieving chromatography separates molecules based on size, and ion-exchange chromatography isolates biologicals based on +ve or -ve charge. Affinity chromatography, on the other hand, purifies a molecule based on its specific function. At present, affinity chromatography with variations is used in purifying a variety of proteins such as receptors, enzymes, antigens, antibodies and several types of recombinant proteins produced by genetic engineering.
However, chromatography requires several buffers. The number and variety of buffers needed in a procedure depends upon the type of components involved and the nature of their interaction. In sieving chromatography, a single buffer is used to eluate proteins from the column. In ion-exchange chromatography, where there is interaction between +ve and -ve charges, a minimum of 2 to 4 types of buffers are required. But, in affinity chromatography, where specific function of a molecule is involved, the variety and number of buffers needed vary considerably. In a typical procedure, an affinity column needs wash buffer, binding buffer, pre-elution buffer, elution buffer, stripping buffer, and storage buffer. The composition, pH and ionic strength of the above mentioned buffers differ considerably from one another. Since their purposes are different, these buffers should not be mixed or contaminated one with the other. Chromatography Buffers Center is ideally suited to hold and supply these differing buffers during the purification of biological molecules.
Technical problems associated particularly with the liquid phase of chromatography and the solutions offered by Chromatography Buffers Center are best explained by taking affinity chromatography as an example. The preferred method of doing this chromatography is by packing affinity beads in a column. Protein solution and various buffers are then passed through. When the volume of protein solution is small, different buffers are added in small aliquots by using a pipette. As the added solution percolates into the beads, more is added. In this approach, every time a buffer is added, the top part of gel bed gets disturbed sending beads floating in the buffer. The floating beads then nonspecifically stick to the wall of column. More the height of added buffer, greater the amount of adsorption of beads to the surface. The adsorbed beads can not be processed since they are not in gel bed. This type of loss of beads results in decreased protein binding capacity of the column. The column adsorbed beads may even contaminate the purified end product by untimely releasing other molecules. This is the first problem.
The volume of added buffer and the amount present on gel bed can be kept low to minimize the loss of beads during the run. However, any delay between additions of buffer makes gel bed go dry, crack and then trap air. A column once dried initiates unpredicted behaviour in the elution pattern of proteins. The end result is getting only a partially pure protein, low yield and highly diluted solution. This is the second problem.
In affinity chromatography, different types of buffers are taken usually in containers such as beakers or flasks. Any mistake in the sequence of addition of buffers from these congregated containers, results in getting less than desired end product. Even, total or partial loss of protein is not uncommon. Because of divided attention between collecting buffer and not allowing gel bed go dry, adding wrong buffer at right time is common. This is the third problem.
Another method of adding buffers to affinity column is by using a peristaltic pump containing a tubing. Keeping the end of tubing always submerged in buffers is very important. The tube is generally attached to the wall of buffer container such as a beaker by an adhesive tape. Because the surface is moist, adhesive tape does not stick strongly and often releases the tubing. The tubing which usually comes as coil, tends to go into coil. This makes the tubing end come out of buffer. If not paid immediate attention, air is pumped into the column which makes the gel bed go dry and crack. This is the fourth problem.
Also, to pump different buffers, the tubing has to be transferred from one buffer container to another. This makes buffers get contaminated either with proteins or with other buffers. When several chromatography runs have to be made, contaminated buffers can cause serious problem in the isolation of a pure protein. This is the fifth problem.
A different approach to adding buffers is by using special equipment. These instruments pump buffers steadily and also switch valves automatically to pump one buffer after another as dictated by a computer. The drawbacks with these instruments, besides prohibitive cost, are leaky valves, need of extra time to warm up and necessity for a lot of buffer for washing and priming pumps and system lines. Maintaining these instruments is costly and time consuming. Some of the affinity chromatography buffers are expensive to waste for washing and priming pumps. Even though, these instruments still need separate containers to hold different types of buffers. In these containers, tubing end surfacing in buffers is a real problem. Therefore, for many laboratories which have limited budget and need, these expensive instruments are least desired. Such labs need alternate instruments. This is the sixth problem.
In brief, keeping gel surface flat with minimum amount of buffer on the top of gel bed, never allowing column go dry, letting the tubing end stay in solution, eliminating cross-contamination of buffers and feeding columns at a steady flow, play important role in obtaining a pure protein.